Iron is an essential micronutrient required for numerous life processes and therefore a rate-limiting growth factor for the proliferation of pathogenic bacteria. As opposed to Fe3+ acquisition systems, there is a fundamental gap in understanding how prokaryotes accumulate Fe2+. Under anoxic conditions, such as that of the GI track where Fe2+ is more abundant than Fe3+, the relatively uncharacterized ferrous iron uptake system, Feo, is responsible for Fe2+ acquisition. The most essential Feo component is FeoB, a transmembrane protein that contains N-terminal GTP-binding and guanine dissociation inhibitor (GDI) domains similar to those found in eukaryotic G-protein coupled receptors and signaling proteins. For this reason, FeoB is speculated to be an ancestral precursor to human G-proteins. Because knockout FeoB bacteria strains have difficulty colonizing GI tracts of mouse models, FeoB represents an attractive antimicrobial target. The ultimate goal of this project is to elucidate the molecular mechanism by which Feo facilities Fe2+ acquisition by prokaryotes. Presently, it is unknown how the Feo system acts to facilitate Fe2+ transport. It is postulated to act either as a GTP-gated ion channel, a GTP-energized active transporter, or a GTP-regulated signaling and Fe2+-sensing protein. To discern among these mechanisms, four specific aims will be pursued. First, the kinetics of the FeoB GTPase activity will be characterized. Then the influence of Fe2+ and a second Feo protein, FeoA, on the FeoB GTPase activity tested. Under the first aim, the function of FeoA will be further defined by examining FeoA- FeoB interactions. Second, in order to distinguish between the proposed signaling and transport mechanisms, Fe2+ uptake assays using FeoB-containing membrane vesicles will be conducted. These same assays will be used to assess the metal ion specificity of FeoB. Third, sequence analysis suggests several conserved transmembrane residues are key for metal ion selectivity and/or communication between the transmembrane and cytosolic regions. Mutagenic studies will be conducted to probe how GTP binding and Fe2+ transport are coupled. Lastly, crystal structures of the cytosolic FeoB domain in its apo, GTP and GDP bound forms will be determined to provide mechanistic insight into GTPase activity and the regulation of FeoB. Taken together, these experiments will shed light on the mechanism of a novel metal transport system used broadly among prokaryotes that is unique to their survival. In addition, the proposed research is expected to advance understanding of metal ion uptake and regulation. PUBLIC HEALTH RELEVANCE: This proposal will examine the molecular mechanisms by which the Feo system facilitates Fe2+ accumulation by prokaryotes under anoxic or reducing conditions, such as those found in the GI tract. Because the Feo system is unique to prokaryotes and vital for their growth, understanding the mechanism that governs Fe2+ uptake processes by Feo will allow for the future development of targeted therapeutic strategies against pathogens.